Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design

Rappuoli, Rino; Bottomley, Matthew J.; D’Oro, Ugo; Finco, Oretta; De Gregorio, Ennio

knowledge of the correlates of vaccine-induced protection or biological signatures of responsiveness are becoming fundamental in the development of novel vaccines. Both memory B cells (MBCs) and plasmablasts (peaking at day 8 after vaccination) have been used to generate naturally derived antigen-specific mAbs. MBCs were shown to be more suitable for this kind of application because of their capability to secrete Abs after EBV immortalization and in the presence of a TLR9 ligand and/or allogeneic irradiated mononuclear cells (Traggiai et al., 2004) . Usually, total peripheral blood lymphocytes or sorted IgG + MBCs are cultured and the released Abs can be screened for antigen specificity and/or functionality. More recently, it has been discovered that even single plasmablasts can be cultured without immortalization, and they can produce sufficient amounts of Abs to allow screening for Ab specificity and function (Jin et al., 2009; Corti et al., 2011b) . This approach has been particularly successful in isolating NAbs from individuals infected by rapidly evolving viral pathogens, leading to the identification of new target molecules that induce the most potent or broadly neutralizing response without prior knowledge of their nature. The power of the characterization of the Abs produced by human B cells that were generated in vivo in response to specific infections has been proved so far for different viruses, such as influenza, HCMV, dengue, and RSV (Beltramello et al., 2010; Corti et al., 2010 Corti et al., , 2013 Macagno et al., 2010; Krause et al., 2011; de Alwis et al., 2012) . Two emblematic examples of how human mAbs can be valuable tools to discover or design new anti-gens come from HCMV and RSV. Most studies on HCMV have focused on the viral glycoproteins gB or gHgL as targets of NAbs. By screening mAbs from an immune donor based on their capacity to neutralize infection in vitro, a panel of potent NAbs was identified. These Abs recognize multiple antigenic sites on a pentameric complex of gH/gL/pUL128/ pUL130/pUL131A, which was not previously known to be the target of NAbs (Macagno et al., 2010) . In a follow-up study, a pentamer-based vaccine was reported to elicit very high NAb titers in mice (Kabanova et al., 2014) , providing evidence of the power of the reverse vaccinology 2.0 approach for the identification and design of improved HCMV vaccine candidates. Furthermore, independent structural and immunological studies have also demonstrated the antigenic potential of the HCMV pentamer in mice (Wen et al., 2014; Ciferri et al., 2015) . A second example comes from the analysis of the NAb response to the F protein of RSV. Initially, the postfusion F protein of RSV was chosen as a candidate to develop a vaccine because it is a very stable protein and is recognized by a neutralizing mAb (palivizumab) that is used to prevent RSV infection in newborns (The IMpact-RSV Study Group, 1998) . However, a promising alternative to the postfusion F antigen has also been identified. RSV-specific human B cells transduced with Bcl-6 and Bcl-xL were used as a source to identify Abs that effectively neutralized the virus. By screening B cell culture supernatants, a NAb called D25-many times more potent than palivizumab-was isolated and shown to completely prevent viral replication in Table 1 . Historical milestones tracking the impact of new technologies on vaccine discovery and design